Effect of micro- and macroencapsulation on oxygen consumption by pancreatic islets.

Immunoisolation of pancreatic islets is extensively investigated for glycemic control in diabetic experimental animals. We previously reported that subcutaneous xenotransplantation of bovine islets protected by a selective polysulfone membrane successfully controlled glycemia in diabetic rats for up to 20 days. We then wondered whether immunoisolated islets have adequate oxygen supply in this device, where only diffusive transport allows cell function and survival. Here we set up an experimental technique to measure oxygen consumption rate (OCR) using a Clark's electrode inserted in a glass thermostated chamber connected to a data recorder and acquisition system. Bovine islets were isolated from 6-month-old calves, encapsulated in sodium alginate microcapsules or inserted in polysulfone hollow fibers. After 1 and 2 days in culture a series of measurements was performed using free islets (at normal or high-glucose concentration), islets encapsulated in microcapsules, or in hollow fibers. In free islets OCR averaged from 2.0 ± 0.8 pmol/IEQ/min at low-glucose concentration and from 2.5 ± 1.0 pmol/IEQ/min at high-glucose concentration ( p < 0.01). OCR in islets encapsulated in microcapsules and in hollow fibers was comparable, and not significantly different from that measured in free islets. Two days after isolation OCR averaged 2.3 ± 0.6 in free islets, 2.3 ± 0.9 in alginate microcapsules, and 2.2 ± 0.7 pmol/IEQ/min in hollow fibers. These results show that OCR by bovine islets is comparable to that previously reported for other species. OCR increases in islets stimulated with high glucose and may be considered as a functional index. Moreover, islet encapsulation in alginate microcapsule, as well as in hollow fiber membranes, did not significantly affect in vitro OCR, suggesting adequate islet oxygenation in these conditions

Effect of inborn pancreatic islet deficit in the Munich Wister Frömter rat

The total mass of pancreatic islet cells is a critical factor in glucose metabolic control. The aim of the present study was to investigate whether in the Munich Wistar Frömter (MWF) rat, beside a reduction in the number of nephrons, there are also alterations in the number of pancreatic islets and of β cell mass. We also examined glucose metabolism, both in normal conditions and following intravenous glucose injection. The number of islets per pancreas, estimated by morphometrical analysis, was significantly lower in MWF rats than in Wistar rats (3,501±1,285 vs. 7,259±2,330 islet/rat, respectively). Also the mean number of islets per gram of body weight was significantly lower in MWF rats than in Wistar rats (18±7 in MWF rats vs. 28±10 islets/g bw in Wistar rats). Morphometric analysis of β cell mass showed an average of 77.1±7% islet cells staining for insulin in MWF rats and 83.9±2.1% in the control Wistar rats. Despite the lower number of islets and β cells, MWF and Wistar rats had comparable fasting blood glucose levels but significant differences in blood glucose following an intraperitoneal glucose tolerance test. In summary, pancreatic islets of MWF and Wistar rats showed a marked difference in morphometrical characteristics. While this difference is not associated with blood glucose levels, glucose metabolism after IPGTT between MWF and Wistar rats is significantly different. These data suggest that an inborn deficit in β cell mass of about 60% is responsible for altered glucose metabolism and could favor the development of diabetes

Differentiation of Mesenchymal Stem Cells towards an insulin-releasing phenotype after co-culture with Pancreatic Islets

Transplantation of pancreatic islets has become a promising clinical option to treat patients with type 1 diabetes, alternative to the standard therapy with insulin injections. Islet transplantation is a minimally invasive therapeutic approach, and it allows a better metabolic control and a long-term insulin independence in more than 80% of patients (Ryan et al., 2002). However this therapeutic treatment has some side effects, such as the poor yield of pancreatic islet explants and even more the immune graft rejection, which have as a consequence the very limited lifespan of transplanted pancreatic islets. To avoid these side effects several strategies have been proposed and, besides the treatment with immunosuppressive drugs, promising results have been obtained with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the survival of many cell types (Scuteri et al., 2006). Several in vivo studies have demonstrated that the concurrent transplantation of pancreatic islets with MSCs reduces the number of islets required to achieve glycemic control in diabetic rats, but the mechanisms of these encouraging results are still unknown (Figliuzzi et al., 2009). For these reasons in this in vitro study we characterized the effect of co-culture of rat MSC on survival and functioning of rat pancreatic islets, by evaluating for 4 weeks: i) MSC adhesion to pancreatic islets; ii) viability of pancreatic islets co-cultured with MSCs; iii) the expression of insulin after co-culture; iv) the ability of co-cultured pancreatic islets to correctly adjust insulin release after variation of glucose concentration. Our results demonstrated that MSCs are able to adhere to pancreatic islets, but to increase only partly the pancreatic islet survival, which retain the ability to express and correctly release insulin after glucose variation in medium culture. Noteworthy that the insulin level in the medium of co-cultured pancreatic islets is always higher with respect to medium of pancreatic islets alone. The immunofluorescence analysis reveals that also MSCs (and not only pancreatic islets) are able to express insulin, but only in co-culture. These results, which justify the in vivo observation reported above, suggest that MSCs undergo to differentiation into a insulin-releasing phenotype after co-culture with pancreatic islets. We are now evaluating the molecular mechanisms which drive this effect, by analyzing the role of soluble factors and of proteins able to induce insulin expression. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001

Can we delay or even reverse diabetic complications by islet transplantation?

Treatment of diabetes is of crucial importance not only for glycemic control but also for the prevention of complications that causes irreversible damage to peripheral nerves, heart, kidney, and eyes. Despite a major effort to obtain prolonged metabolic control, diabetes complication can only be delayed in time. In a recent publication we investigated the effect of islet transplantation on diabetic complications in a rat model of streptozotocin-induced diabetes (1). We reported that islet transplantation induced an amelioration of impaired nociceptive threshold and normalization of nerve conduction velocity in diabetic rats. Cardiomyocyte loss was also reduced by islet transplantation and the observed mild kidney damage of diabetic rats was prevented. We concluded that transplantation of pancreatic islets, besides controlling glycemia, induced regression of neuropathy and prevented cardiovascular alterations, which was always there supporting the potential of islet transplantation. In this review we compare our recently published data with previous reports on prevention and regression of diabetic neuropathy, cardiovascular disease, nephropathy, and retinopathy with a particular attention to those on animal models of diabetes. In details, the potential of islet transplantation in inducing the regression of diabetic complications is discussed

Toxicological evaluation of airborne particulate matter. Are cell culture technologies ready to replace animal testing?

Exposure to atmospheric particulate matter (PM) can affect human health, causing asthma, atherosclerosis, renal disease and cancer. In the last few years, outdoor air pollution has increased globally, leading to a public health emergency. Epidemiological studies have reported a correlation between the development of severe respiratory and systemic diseases and exposure to PM. To evaluate the toxic effect of PM of different origins, conventional experimental toxicological investigations have been conducted in animals; however, animal experimentation poses major ethical issues and usually differs from human conditions. As an alternative, human cell cultures are increasingly being used to investigate cellular and molecular mechanisms of PM toxicity. Although 2D cell cultures have been proven helpful, they are far from being a valid alternative to animal tests. Recently, 3D cell culture and organ-on-chip technology have provided systems that are more complex and that can be more informative for toxicity studies. In this review, the results of the 2D systems that are most frequently used for PM toxicity evaluations are summarized with a special focus on their limitations. We also examined to which extent 3D cell culture and particularly the organ-on-chip technology may overcome these limitations and represent effective tools to improve airborne PM toxicity evaluations

Effect of the 3D Artificial Nichoid on the Morphology and Mechanobiological Response of Mesenchymal Stem Cells Cultured In Vitro

Stem cell fate and behavior are affected by the bidirectional communication of cells and their local microenvironment (the stem cell niche), which includes biochemical cues, as well as physical and mechanical factors. Stem cells are normally cultured in conventional two-dimensional monolayer, with a mechanical environment very different from the physiological one. Here, we compare culture of rat mesenchymal stem cells on flat culture supports and in the &ldquo;Nichoid&rdquo;, an innovative three-dimensional substrate micro-engineered to recapitulate the architecture of the physiological niche in vitro. Two versions of the culture substrates Nichoid (single-layered or &ldquo;2D Nichoid&rdquo; and multi-layered or &ldquo;3D Nichoid&rdquo;) were fabricated via two-photon laser polymerization in a biocompatible hybrid organic-inorganic photoresist (SZ2080). Mesenchymal stem cells, isolated from rat bone marrow, were seeded on flat substrates and on 2D and 3D Nichoid substrates and maintained in culture up to 2 weeks. During cell culture, we evaluated cell morphology, proliferation, cell motility and the expression of a panel of 89 mesenchymal stem cells&rsquo; specific genes, as well as intracellular structures organization. Our results show that mesenchymal stem cells adhered and grew in the 3D Nichoid with a comparable proliferation rate as compared to flat substrates. After seeding on flat substrates, cells displayed large and spread nucleus and cytoplasm, while cells cultured in the 3D Nichoid were spatially organized in three dimensions, with smaller and spherical nuclei. Gene expression analysis revealed the upregulation of genes related to stemness and to mesenchymal stem cells&rsquo; features in Nichoid-cultured cells, as compared to flat substrates. The observed changes in cytoskeletal organization of cells cultured on 3D Nichoids were also responsible for a different localization of the mechanotransducer transcription factor YAP, with an increase of the cytoplasmic retention in cells cultured in the 3D Nichoid. This difference could be explained by alterations in the import of transcription factors inside the nucleus due to the observed decrease of mean nuclear pore diameter, by transmission electron microscopy. Our data show that 3D distribution of cell volume has a profound effect on mesenchymal stem cells structure and on their mechanobiological response, and highlight the potential use of the 3D Nichoid substrate to strengthen the potential effects of MSC in vitro and in vivo